Evidence from heterologous expression of glutathione S-transferases A and A1 of the plaice (Pleuronectes platessa) that their endogenous role is in detoxification of lipid peroxidation products

cDNA clones for glutathione S-transferases A (GST-A) and A1 (GST-A1) from plaice (Pleuronectes platessa) were expressed as N-terminally 6XHis tagged proteins in Escherichia coli and purified to homogeneity from Ni-NTA silica. GST-A was an efficient catalyst for conjugation of unsaturated alkenals derived from peroxidation of polyunsaturated fatty acids with the highest activity observed with trans-non-2-enal (8 micromol min(-1) mg(-1)). GST-A1 was a very efficient Se-independent glutathione peroxidase with an activity towards cumene hydroperoxide of 25 micromol min(-1) mg(-1). Although the enzymes exhibited moderately high activities towards the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) they exhibited little or no activity towards other common prototypical xenobiotic substrates. Together with data for ontogeny, tissue distribution and inducibility of these enzymes, we contend that a primary function of these enzymes is protection from the harmful effects of lipid peroxidation products generated naturally or exacerbated by xenobiotic exposure.     

金属蛋白酶对大菱鲆血清抗氧化酶活性及丙二醛含量的影响

为了研究金属蛋白酶对大菱鲆(Scophthalmus maximus)抗氧化系统的影响,作者分别对大菱鲆肌肉注射不同浓度的金属蛋白酶,并于3、6、12、24、48、72 h测定血清中谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、总超氧化物歧化酶(T-SOD)、总抗氧化活力(T-AOC)以及丙二醛(MDA)含量变化。结果显示,低质量浓度组(0.037mg/mL)金属蛋白酶处理后,CAT和T-SOD活力除3h显著高于对照组外(P0.05),其余时间点均与对照组无显著差异(P0.05),而低浓度组GSH-Px和T-AOC在整个试验过程中均与对照组无显著性差异(P0.05)。中质量浓度组(0.073mg/mL)金属蛋白酶胁迫后血清中CAT、GSH-Px、T-SOD和T-AOC在3 h～72 h均呈现波浪形的变化趋势。高质量浓度组(0.147 mg/mL)金属蛋白酶对上述4种抗氧化酶产生不同程度的抑制,且在6 h～72 h均显著低于对照组(P0.05)。金属蛋白酶低质量浓度和中质量浓度组MDA含量呈现较为平稳的变化趋势,且与对照组无显著性差异(P0.05),而高质量浓度组MDA含量在3 h～72 h均显著高于对照组(P0.05)。     

The effect of long-term depuration on levels of oxidative stress biomarkers in mullets (Mugil cephalus) chronically exposed to contaminants

The present study aimed to obtain additional data on the effect of long-term depuration on the levels of oxidative stress biomarkers, and to clarify the role of mullets for monitoring pollution in River Douro estuary. Mullets chronically exposed to a mixture of contaminants in Douro estuary were captured in Spring of 2001, 2002 and 2003. The activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX); and oxidative damages in lipids (lipid peroxidation) and in proteins (protein carbonyl content) were assessed at capture day and after transfer to unpolluted seawater for 1, 4 and 8 months. An overall decrease in the activities of the antioxidant enzymes was detected, except for the GPX after 4 months depuration. CAT activity exhibited the more significant decrease at the end of the long-term depuration. The decrease in SOD activity after 1 month of depuration was then maintained during the remaining depuration period. Regarding oxidative damages, a decrease in lipid peroxidation as well as the content of oxidised proteins was observed during depuration. Indeed, at capture the activities of antioxidant defences were higher as a result of the formation of reactive oxygen species (ROS) from the metabolism of pollutants. The oxidative damaged molecules were repaired or degraded during the depuration period, supporting the use of such damages as indicators of exposure to pro-oxidant pollutants.     

大菱鲆卵子排放后在卵巢腔内老化特征初探

为了探明大菱鲆(Scophthalmus maximus)卵子在卵巢腔内老化的原因及特征,采用连续跟踪采卵的方式,通过荧光染色、生理生化指标测定,对不同采卵时间(0, 3, 6, 9, 12 h)的卵子活性氧和脂质过氧化情况(氧化产物丙二醛MDA)、抗氧化系统(超氧化物歧化酶SOD,还原型谷胱甘肽GSH)、卵巢液pH值及受精率和孵化率进行对比研究。结果表明,随着卵子在体内老化时间的延长,其活性氧含量及脂质过氧化程度逐渐升高,活性氧含量及过氧化程度与排卵后卵子老化呈正相关;SOD酶活力,GSH酶活力均显著下降,MDA含量显著提高;受精率和孵化率均显著下降。排卵后6h,孵化率降到2.87%～10.60%,此时的卵质已经极度恶化,不能再继续用于后续的生产中。综上,在卵巢腔中,卵子排放后,随着时间的延长,活性氧含量显著升高,抗氧化能力逐渐下降,受精率和孵化率显著降低,老化逐渐加剧。     

军曹鱼(Rachycentron canadum)幼鱼对环境低氧胁迫氧化应激与能量利用指标的响应

本文探究环境低氧对军曹鱼(Rachycentron canadum)氧化应激和能量利用指标的影响,为军曹鱼的健康养殖提供参考依据。通过设置低氧胁迫–恢复实验,将军曹鱼幼鱼(平均体质量(220.67±20.73)g)在低氧((2.64±0.25)mg/L)胁迫3 h及复氧((6.34±0.15)mg/L)8 h、24 h和48 h后,测定其肝脏和肌肉组织的氧化应激与能量利用指标。结果显示,低氧胁迫后,肝脏中丙二醛(Malondialdehyde,MDA)、过氧化氢酶(Catalase,CAT)和谷胱甘肽还原酶(Glutathione Reductase,GR)活力均显著低于对照组(p<0.05),乳酸脱氢酶(Lactate Dehydrogenase,LDH)活性显著高于对照组(p <0.05);肌肉中MDA和脂质过氧化物(Lipid Peroxidase,LPO)活性均显著低于对照组(p<0.05),超氧化物歧化酶(Superoxide Dismutase,SOD)和LDH活性均显著高于对照组(p<0.05);肌糖原和肝糖原含量极显著低于对照组(p<0.0...     

红树叶片衰老过程中活性氧和保护酶活性的变化

测定了红树植物木榄(Bruguiera gymnorrhiza)和秋茄(Kandelia candel)幼叶、成熟叶和老叶间活性氧和三种细胞保护酶活性的变化。结果表明,在叶片衰老过程中,丙二醛和H2O2含量显著提高,超氧化物自由基产生速率加快,表明红树叶片衰老中膜脂过氧化作用增强,并且与H2O2和超氧化物自由基含量的上升相关。在3种细胞保护酶中,超氧化物歧化酶(SOD)活性逐渐降低,过氧化物酶(POD)活性显著提高,过氧化氢酶(CAT)活性在叶片成熟过程中增高,随着衰老而急剧降低,细胞保护酶酶活的变化直接影响了叶片衰老过程中活性氧代谢的平衡。     

水体Hg^2＋对中华绒螯蟹（Eriocheir sinensis）血淋巴抗氧化酶活力和丙二醛含量的影响

采用单因子实验设计方法,进行了水体汞（Hg^2＋）在不同暴露时间对中华绒螯蟹血淋巴超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GPX）和过氧化氢酶（CAT）活力以及脂质过氧化产物丙二醛（MDA）含量的影响研究。结果表明,较低浓度Hg^2＋（0．01和0．05mg／L）处理组中华绒螯蟹血淋巴SOD、GPX、CAT活力在...     

3种海洋赤潮微藻抗氧化酶活性对UV-B辐射增强的响应

利用生物化学的方法,研究了赤潮异弯藻、亚历山大藻和中肋骨条藻抗氧化酶活性对UV-B辐射增强的响应变化。结果表明:(1)不同赤潮微藻的抗氧化酶活性对UV-B辐射增强的响应变化是不同的。(2)不同的抗氧化酶活性对UV-B辐射增强的响应变化同样存在明显的差异。(3)与过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)相比,超氧化物歧化酶(SOD)活性对UV-B辐射增强的响应最敏感,谷胱甘肽过氧化物酶(GPX)活性的响应变化最不明显。3种赤潮微藻SOD活性的变化是指示UV-B辐射增强的优选生物学指标。     

肿瘤放射治疗中扇贝硒蛋白的作用

以雄性昆明种小鼠为实验对象,在接种瘤株之前ｄ,放疗实验组以及扇贝硒蛋白（Ｓｅｌｅｎｉｕｍ－ＰｒｏｔｅｉｎｏｆＣｈｌａｍｙｓｆａｒｒｅｒｉ（ＳＰＣＦ）胃饲,接种Ｓ１８０瘤株后,继续胃饲,连续２２ｄ,结果表明,扇贝硒蛋白使肿瘤生长减缓,并使荷瘤鼠因放疗而下降的ＧＳＨ－Ｐｘ（Ｇｌｕｔａｔｈｉｏｎｅｐｅｒｏｘｉｄａｓｅ）,ＳＯＤ（Ｓｕｐｅｒｏｘｉｄｅｄｉｓｍｎｔｕｓｅ）ＣＡＴ酶活性增强,ＬＰＯ（Ｌｉｐｉｄ     

Anthropogenic pollution stimulates oxidative stress in soft tissues of mussel <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (Dunker1853)

The digestive gland and gills of the mussel Crenomytilus grayanus extracted from three locations — (i) sampled from a clean and (ii) polluted site and (iii) transplanted from the nonpolluted to polluted site - were analysed for antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase), total oxyradical scavenging capacity and levels of lipid peroxidation products (malondialdehyde, conjugated dienes and lipofuscin). Perturbation of redox status was found in both digestive gland and gill tissues of mussels living in the polluted area. As the activities of superoxide dismutase and catalase were 1.2–3 times higher, the total oxyradical scavenging capacity was lower by 20–35% and the levels of lipid peroxidation products were 2–7 times higher compared to mussels from the reference site. In transplanted mussels, the lipid peroxidation process in both tissues was significantly stimulated (the level of conjugated dienes was increased 1.7–2.5-fold; malondialdehyde and lipofuscin contents were increased 3.5–5-fold) and the total oxyradical scavenging capacity fell by 50–70%. In addition, the transplantation generally resulted in transient and variable responses of antioxidant enzymes for both tissues. Complex response-behaviour of the antioxidant enzymes strongly points to the necessity of employing a combined approach that takes into account activities of antioxidant enzymes and the total oxyradical scavenging capacity, as well as measurement of oxidative damage (e.g., lipid peroxidation) to evaluate the physiological health of molluscs.     

哈维氏弧菌对条纹斑竹鲨4种酶活性的影响

以哈维氏弧菌（Vibrio harveyi）对条纹斑竹鲨（Chiloscyllium plagiosum）进行病原接种试验,分别在4、8、12、24、48、72、96h后测定肝脏、脾脏、鳃及血清的酸性磷酸酶（acid phosphatase,ACP）、碱性磷酸酶（alkaline phosphatase,AKP）、超氧化物歧化酶（superoxide dismutase,SOD）和血清中溶菌酶（lysozyme,LSZ）的活力.实验结果表明：对照组条纹斑竹鲨的这4种酶的活力呈现出明显的组织差异性,血清中SOD活力为124.32 U/cm3,显著高于其他3种酶的活力;其他组织中3种酶的活力由高到低依次为SOD、ACP、AKP.在实验组条纹斑竹鲨被感染4～72h期间,血清中SOD活力明显下降,ACP活力持续下降,感染96h后除LSZ外,其他3种酶的活力都呈回升趋势;其他组织中ACP与AKP活力变化均为被感染4h后下降,12h后明显回升,SOD则在被感染4h后活力上升,而后下降,96h后回升.这4种酶的活力的变化主要是应激作用与非特异性免疫防御作用共同作用导致的结果.     

中华哲水蚤抗氧化系统对海水酸化的响应

海洋浮游动物可以对因二氧化碳浓度升高导致的海水酸化做出敏感的响应,然而关于海水酸化对其生理机能的研究却较少。因此,我们研究了暴露于不同二氧化碳浓度（0.08,0.20,0.50 and 1.00%）的酸化条件下中华哲水蚤主要抗氧化酶以及2种解毒酶的活性变化情况。结果表明,暴露于酸化海水中的中华哲水蚤的GPx酶活性显著高于对照组,然而,其它抗氧化组分,包括GST、SOD活性,GSH水平和GSH/GSSG比值均被显著抑制。中华哲水蚤的ATPase活性被海水酸化显著刺激,而AchE活性却显著被抑制。此外,主成分分析结果表明,各指标变化的75.93%可以用第一和第二主成分解释。因二氧化碳浓度升高导致的海水酸化可以通过影响桡足类体内一些酶类的活性来影响其新陈代谢及存活。在未来的研究工作中,需要对海水酸化和其它环境因素的协同作用对桡足类的影响作进一步研究。     

Effect of seasonality on oxidative stress responses and metal accumulation in soft tissues of Aulacomya atra,a mussel from the South Atlantic Patagonian coast

This study investigated the effects of pollution and its interaction with temperature on the oxidative status of the ribbed mussel Aulacomya atra in the southern Atlantic Patagonian coast. Animals were collected from four sites with different degree and type of human activity impact, during the summer and winter of 2011. Seawater chromium, copper, manganese, nickel and zinc concentrations were measured, as well as metal accumulation, lipid peroxidation, protein oxidation, reduced glutathione levels, and enzymatic activities of superoxide dismutase and glutathione-S-transferase in gills and digestive glands.Metal bioaccumulation and oxidative stress responses in both tissues were generally higher in mussels from harbor areas. Water temperature had a remarkable effect on gill SOD activity and protein oxidation during winter in mussels from all locations.Methodologically, we conclude that measuring both metal bioaccumulation and oxidative stress responses allowed for a more accurate assessment of the biological effects of metal present in seawater.     

Mixed-function oxidase activity and conjugating enzymes in two species of antarctic fish

Enzyme activities of phase I and phase II of the hepatic detoxication system were investigated in two species of fish from the Ross Sea in Antarctica. The fish had extremely low phase I enzyme (BPMO and aldrin epoxidase). The reductase regenerating enzyme activities were comparable to those found in fish of temperate seas. Transferases of cytosol showed highest activity in the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and little or no conjugation of other substrates. The GSSG reductase and the glutathione transferase with peroxidase activity (CuOOH substrate) were low; the Sedependent glutathione peroxidase activity was not detectable. Interspecific differences were found for most of the enzymes tested.     

苯并[a]芘(BaP)对褐菖鲉(Sebasticus marmoratus)肝CYP1A1酶活性、基因表达及蛋白表达的影响

为了了解苯并[a]芘(BaP)对鱼类细胞色素P4501A1(CYP1A1)表达的影响,以褐菖鲉(Sebasticus marmoratus)为实验材料,采用体内实验,研究其在经过不同浓度(0.1、1、10、20、50mg/kg鱼体重量)的BaP诱导后,鱼体肝脏研究CYP1A1基因表达的情况,筛选出后续时间-效应实验中BaP注射的最佳浓度,研究BaP诱导6h、12h、1d、3d、7d后(质量浓度为20mg/kg鱼体重量)鱼体肝脏CYP1A1酶活性、基因表达和蛋白表达的情况。结果表明:剂量-效应实验中,20mg/kg鱼体重量为最佳浓度,此浓度下,基因表达在各组中变化最显著。时间-效应实验中,较空白对照组而言,染毒6h、12h和1d后,EROD酶活性显著增加。3d后开始下降,与对照组相比变化不大,7d后酶活性又发生上调。半定量RT-PCR结果表明,各染毒组与对照组相比,CYP1A1基因表达量都发生了上调,呈现先上升后下降的趋势。其中,6h和12h组相对表达量极显著增加,1d后开始下降且与3d和7d组相比变化不明显。Western blot结果表明,蛋白表达量在染毒12h后表现出显著的诱导效应,随着时间的延长略有回落,但与对照组相比仍有显著性差异。研究表明:BaP对褐菖鲉CYP1A1具有较强的诱导作用。一定质量浓度的BaP注射于褐菖鲉不同的时间后,能诱导褐菖鲉活体EROD酶活性、CYP1A1基因m RNA表达及蛋白表达,并随着时间的延长呈现先诱导后抑制的趋势。这说明BaP作为诱导剂对CYP1A1酶活性和蛋白表达的作用机制可能与调控CYP1A1的转录水平有关。     

Effect of different wavelengths of light on the antioxidant and immunity status of juvenile rock bream, <Emphasis Type="Italic">Oplegnathus fasciatus</Emphasis>, exposed to thermal stress

We investigated the effect of light wavelengths on antioxidant and immunity parameters in juvenile rock bream, Oplegnathus fasciatus, exposed to thermal stress (25 and 30°C). We exposed the fish to light emitting diodes (LEDs) emitting green (520 nm) and red light (630 nm) of 0.25 and 0.5 W/m² intensity, and measured the activity, and mRNA and protein expression levels of the antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase. We also determined the levels of plasma hydrogen peroxide (H₂O₂), melatonin, and lysozyme. Furthermore, the mRNA and protein levels of caspase-3 were measured and terminal transferase dUTP nick end labeling (TUNEL) assays were performed. We observed that mRNA expression and activities of antioxidant enzymes and plasma H₂O₂ levels were significantly higher after exposure to high temperatures. However, increases in these parameters were significantly lower after exposure to green LED light. The plasma melatonin and lysozyme levels were significantly lower in the different groups after exposure to high temperatures; however, in groups exposed to green LED light, their levels were significantly higher than those in the control group. The expression pattern of caspase-3 mRNA was similar to that of H₂O₂. The TUNEL assay showed that apoptosis was markedly higher at higher water temperatures than that at 20°C. These results indicate that high water temperatures induce oxidative stress and decrease the immunity in juvenile rock bream but green LED light inhibits the rise in oxidative stress and combats the decrease in immunity and should, thus, be useful in the culture of rock bream.     

氨氮胁迫对方斑东风螺六种免疫酶活性的影响

本文研究了氨氮对方斑东风螺(Babylonia areolata)的急性毒性及不同浓度的氨氮溶液(22、47.5、102和220 mg/L)对其体内六种免疫酶过氧化氢酶(CAT)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、总超氧化物歧化酶(T-SOD)、谷胱甘肽过氧化物酶(GSH-PX)和过氧化物酶(POD)活性的影响。实验结果表明:氨氮浓度越大,其毒性作用越强,方斑东风螺死亡率越高;500mg/L氨氮处理组于24h便出现(20.3±2.1)%死亡率,并且96h后存活率降为0。而相同条件下,随试验时间的延长,方斑东风螺死亡率也越高。氨氮浓度和处理时间均对方斑东风螺免疫酶活力有显著的影响(P 0.05)。与对照组相比,处理组CAT和AKP活性均表现出"诱导-抑制"的趋势, ACP和T-SOD活性表现出"抑制-诱导"的趋势, CSH-PX活性呈现"诱导-抑制-诱导"的趋势,而氨氮对处理个体POD活性的影响整体表现为抑制作用。可见,养殖水体中的氨氮会对方斑东风螺免疫酶活性产生较大的影响。本研究从生态现象和生理指标相结合的方式阐述了氨氮对方斑东风螺个体的致毒特性,将对方斑东风螺的养殖具有实用意义,同时丰富了氨氮对贝类毒理实验免疫性能层面的基础数据,并为其他贝类氨氮胁迫实验提供参考。     

Oxidative stress in two populations of killifish (Fundulus heteroclitus) with differing contaminant exposure histories

A population of killifish (Fundulus heteroclitus) inhabiting a creosote-polluted inlet of the Elizabeth River demonstrates tolerance to the acute toxic effects exerted by contaminated sediments on reference site killifish. Previous data have suggested that upregulated antioxidant defenses contribute to short-term tolerance in killifish exposed to Elizabeth River sediments. This study investigated population differences in antioxidant defenses from wild caught Elizabeth River and reference population killifish in different seasons, and after being held in the laboratory. Parameters measured in the killifish were total glutathione concentrations (GSH(T)), activities of glutathione reductase (GR), glutathione peroxidase (GPx), and lipid peroxidation (LPO), all in adult hepatic tissues. The Elizabeth River population exhibited greater GSH(T), higher GPx activities, and increased LPO as compared to the reference population. Sex specific population differences were also observed in GSH(T) and GPx. Both populations displayed decreased GSH(T) and increased GR from early to late summer, as well as after being held in the laboratory. This study indicates that there are many factors that may contribute to differences in levels of antioxidant defenses in addition to exposure to contaminants, including reproductive status and environmental conditions.     

Effect of cadmium, copper and mercury on antioxidant enzyme activities and lipid peroxidation in the gills of the hydrothermal vent mussel Bathymodiolus azoricus

Metals are known to influence lipid peroxidation and oxidative status of marine organisms. Hydrothermal vent mussels Bathymodiolus azoricus live in deep-sea environments with anomalous conditions, including high metal concentrations. Although B. azoricus are aerobic organisms they possess abundant methano and thioautotrophic symbiotic bacteria in the gills. The enzymatic defences (superoxide dismutase (SOD), catalase (CAT), total glutathione peroxidase (Total GPx) and selenium-dependent glutathione peroxidase (Se–GPx)) and lipid peroxidation were determined in the gills of B. azoricus exposed to Cd (0.9 μM), Cu (0.4 μM) and Hg (0.1 μM) with different times of exposure. The experiments were performed in pressurized containers at 9 ± 1 °C and 85 bars.Results show that vent mussels possess antioxidant enzymatic protection in the gills. Cd and Cu had an inhibitory effect in the enzymatic defence system, contrarily to Hg. These enzymatic systems are not completely understood in the B. azoricus, since reactive oxygen species might be produced through other processes than natural redox cycling, due to hydrogen sulphide and oxygen content present. Also the symbiotic bacteria may play an important contribution in the antioxidant protection of the gills.     

Effect of cadmium, copper and mercury on antioxidant enzyme activities and lipid peroxidation in the gills of the hydrothermal vent mussel Bathymodiolus azoricus

Metals are known to influence lipid peroxidation and oxidative status of marine organisms. Hydrothermal vent mussels Bathymodiolus azoricus live in deep-sea environments with anomalous conditions, including high metal concentrations. Although B. azoricus are aerobic organisms they possess abundant methano and thioautotrophic symbiotic bacteria in the gills. The enzymatic defences (superoxide dismutase (SOD), catalase (CAT), total glutathione peroxidase (Total GPx) and selenium-dependent glutathione peroxidase (Se-GPx)) and lipid peroxidation were determined in the gills of B. azoricus exposed to Cd (0.9 microM), Cu (0.4 microM) and Hg (0.1 microM) with different times of exposure. The experiments were performed in pressurized containers at 9+/-1 degrees C and 85 bars. Results show that vent mussels possess antioxidant enzymatic protection in the gills. Cd and Cu had an inhibitory effect in the enzymatic defence system, contrarily to Hg. These enzymatic systems are not completely understood in the B. azoricus, since reactive oxygen species might be produced through other processes than natural redox cycling, due to hydrogen sulphide and oxygen content present. Also the symbiotic bacteria may play an important contribution in the antioxidant protection of the gills.